Abstract: The human intestinal microbiota is a genetically diverse community of microorganisms that performs critical functions for the host, including carbohydrate metabolism, vitamin production, modulation of the immune system, promotion of epithelial barrier function, and exclusion of pathogens. Disruption of the microbiota can lead to a number of pathologies in the host, and probiotics have emerged as a potential solution for treating or preventing such dysbioses. Probiotic treatments consist of the consumption of a strain or several strains of live microorganisms that confer benefits on their human hosts when administered in adequate concentrations. We propose a novel synthetic biology approach to address the most critical barrier currently facing probiotic therapeutic efficacy, namely the inability of probiotic bacteriato permanently and stably integrate into established human microbiota. We hypothesize that (1) native human microbiota, either during development or when stabilized, encode a repertoire of genes which would enable stable integration into an established community and (2) these traits can be engineered into organisms with putative probiotic properties, improving their intestinal colonization potential and fitness. Accordingly, we will functionally prospect complete human microbiota and defined gut microbial culture collections for genes and operons facilitating integration (collectively referred to as integration-enhancing fragments or IEFs) of a set of candidate probiotics into stable microbiota of mammalian hosts, investigate the mechanisms of the identified genes, and compete and combinatorially optimize the best IEFs to select and engineer enhanced integration phenotypes. We will also confirm the efficacy of our enhanced probiotics by comparing them to traditional probiotics in a mouse model for intestinal disease. To achieve these goals, we will develop and apply cutting- edge technological innovations in functional metagenomics, next-generation sequencing and gnotobiotic husbandry. Hence, a major anticipated deliverable of our proposal is a novel platform technology for engineering fitness-enhancing properties into putative probiotic bacteria, promoting integration and persistence in a developed and stable microbiota in a mammalian host. We will apply this platform to engineer next-generation probiotic strains with an enhanced capacity for integration into an established microbiota, with the potential for rapid therapeutic deployment for the treatment of human gastrointestinal diseases. Public Health Relevance: Modulation of the human gastrointestinal microbiota through probiotic therapy is being considered for the treatment of a number of gastrointestinal disorders. This proposal aims to address one of the most critical barriers to the deployment of current putative probiotic strains, namely their inability for long-term integration into the host microbioa. We will develop novel technologies to discover and engineer genetic elements which enable candidate probiotic strains to stably integrate into established microbiota, with the potential for rapid deployment for the treatment of human gastrointestinal disease.